| BackgroundIn accordance with a report by the World Health Organization,an estimated 180 000 deaths every year are caused by burns,and the vast majority occur in low-and-middleincome countries(https://www.who.int/news-room/fact-sheets/detail/burns).The physiological and pathological responses of burns are more complex than other types of trauma,with characteristics that include more inactivated tissue,more exudation,more susceptible to infection,and easier to leave scars.The healing process of burn wounds consists of four interrelated,interacting,and overlapping phases: hemostasis,inflammation,proliferation,and remodeling.Wound repair and functional reconstruction of skin tissue are based on the coordinated advancement of these four phases,which complement each other.During this process,any unfavorable factors such as persistent inflammation,infection,microenvironmental imbalance,etc.will affect the healing process,leading to delayed or non-healing,scar formation,tissue contracture and other adverse healing outcomes,which will affect the patient’s quality of life in the long term.Currently,for the treatment of deep burn wounds,surgical excision of necrotic tissue followed by autologous skin grafting to repair wounds or temporary covering and protection of wounds with allogeneic skin is the "gold standard" for repairing skin defects,but there are some obvious problems,such as damage to the donor skin area,insufficient skin source,immune rejection and the risk of spreading viral diseases.In recent years,with the development and innovation of stem cell therapy,biomaterials and nanotechnology,tissueengineered skin,as a new type of skin substitute,provides a new therapeutic idea to realize permanent alternative treatment for skin defects.Tissue engineered skin is mainly composed of seed cells,bioactive factors and scaffold materials.Mesenchymal stem cells(MSC)have been widely studied and used in the construction of tissue engineered skin due to their role in reducing inflammation,promoting angiogenesis and wound repair;the properties of human amniotic membrane tissue in promoting wound healing,tissue reconstruction,regulating inflammation and inhibiting microorganisms have been recognized by a large number of preclinical studies or clinical trials;microcarrier technology,as a new method of cell cultivation and expansion,has provided a new direction to solve the problem of the long in vitro cultivation time of stem cells and the high risk of differentiation;in-situ forming hydrogel as a new type of wound dressing,has been widely studied in the field of tissue engineering to promote wound healing.Based on the above research background,MSC,human amniotic membrane tissue and in-situ forming hydrogel have shown great potential in the field of wound repair,which can help the construction of multifunctional wound repair materials and provide new solutions for the treatment of burns and other wounds.ObjectivesBased on the concept and technology of tissue-engineered skin,we proposed to construct a multifunctional and integrated wound repair material using MSC,human amniotic membrane tissue and in-situ forming hydrogel to achieve a synergistic therapeutic regimen in which stem cell therapy and hydrogel scaffolds complement each other’s functions in order to alleviate inflammatory response,promote vascularization,and accelerate healing of burn wounds.Part Ⅰ: Human amniotic membrane tissue was used to prepare mAM(micronized amnion membrane),which was used as a microcarrier and combined with a 3D culture system to culture and expand umbilical cord-derived MSC(UC-MSC)in vitro to construct mAM-MSC,and mAM-MSC were further used as seed cells to promote burn wound healing.mAM-MSC samples were taken from the 3D culture system at regular intervals to observe the survival of UC-MSC on mAM;to detect the efficiency of mAM in expanding MSC in vitro;to compare the differences in gene expression of MSC under 2D and 3D culture conditions;to detect the effects of mAM on the biological functions(paracrine,angiogenesis,and wound repair)of MSC and to explore the potential mechanisms.Part Ⅱ: Preparation of in-situ forming CPOD hydrogel(CMCS-PVA-OHA-DA hydrogel)using carboxymethyl chitosan(CMCS),polyvinyl alcohol(PVA),oxidized hyaluronic acid(OHA),and dopamine(DA);observation of the characterization,mechanical properties,tissue adhesion,antimicrobial and antioxidant abilities of CPOD hydrogel and investigation of the potential mechanisms.Part Ⅲ: Based on the concept of tissue-engineered skin,mAM-MSC and CPOD hydrogel from the first two parts of the study were combined and used as seed cells and scaffolds for the treatment of burn wounds,respectively.Firstly,in vitro experiments were carried out to investigate the effects of mAM-MSC composite CPOD hydrogel on pivotal cells participating in wound repair(macrophages,vascular endothelial cells,fibroblasts);then,in vivo experiments were carried out to observe the regulatory effects of mAM-MSC composite CPOD hydrogel transplantation on the healing of burn wounds;and finally,we explored the mechanism of mAM-MSC composite CPOD hydrogel transplantation in regulating the healing of burn wounds.MethodsPart Ⅰ:(1)Amniotic membrane and umbilical cord tissues from healthy Caesarean section women were obtained through the approval of the Ethics Committee of Shanghai Changhai Hospital and informed consent from all donors;(2)Referring to the previous research of our group,human amniotic membrane tissues were prepared into mAM,and at the same time,UC-MSC were extracted from umbilical cords;(3)mAM was used as microcarrier to culture and amplify the UC-MSC in the 3D culture system,and mAM-MSC was obtained;(4)the microscopic morphology of mAM-MSC was observed by scanning electron microscopy and cytoskeletal staining experiments;(5)the biocompatibility of mAM and the efficiency of MSC amplification in vitro were evaluated by CCK-8 proliferation experiments and live/dead staining;(6)the total RNA from the 2D-cultured MSC and the MSC from mAM-MSC was extracted and subjected to RNA-sequencing(RNA-seq)to explore the biological effects and mechanisms of mAM on MSC;(7)The results of transcriptome sequencing were validated using real-time fluorescence quantitative polymerase chain reaction(RT-q PCR).Part Ⅱ:(1)CMCS and PVA were formulated into CMCS-PVA solution,while OHA and DA were formulated into OHA-DA solution,and the two solutions were connected by a double-barrel syringe with a tee tube,and the feeding rate of CMCS-PVA solution was twice as fast as that of OHA-DA solution,so that it could be cross-linking at the injection site to form the CPOD hydrogel;(2)The CPOD hydrogel was detected by rheology;(3)The microstructure of the CPOD hydrogel was observed by field emission scanning electron microscopy(FESEM);(4)Pigskin was selected for lap shear strength test and wound-closure adhesion strength test to detect the tissue adhesion;(5)The mechanism of tissue adhesion was investigated by X-ray photoelectron spectrometry(XPS);(6)The compression test was used for mechanical performance evaluation;(7)The antimicrobial capacity of the CPOD hydrogel against Escherichia coli and Staphylococcus aureus was detected by plate colonycounting method;(8)The cytocompatibility of CPOD hydrogel was examined by CCK-8proliferation assay and live/dead staining assay;(9)The CPOD hydrogel was embedded in the subcutaneous area of the back of C57BL/6 mice,and the blood was drawn 28 days later to perform the blood routine test and liver and kidney functions tests for further observation of biocompatibility;(10)The antioxidant properties of CPOD hydrogel were observed in vitro using the scavenging assay of free radicals(DPPH,ABTS,and PITO);(11)The effect of CPOD hydrogel in scavenging intracellular reactive oxygen species(ROS)was observed using intracellular ROS staining assay.Part Ⅲ:(1)Detect the effect of mAM-MSC composite CPOD hydrogel on the phenotypic transformation of macrophage(RAW264.7)using flow cytometry to observe its anti-inflammatory ability;(2)Evaluate the biological effect of mAM-MSC composite CPOD hydrogel on vascular endothelial cells(HUVECs)using the Transwell cell migration and tube formation assay to detect the angiogenic function;(3)The effect of mAM-MSC composite CPOD hydrogel on the migration of fibroblasts(HFBs)was observed using the scratch assay to detect its ability to promote wound repair;(4)The C57BL/6 mouse deep second-degree burn wound model was used to observe its effect in burn wound healing.The mice were divided into sham group,mAM-MSC group,CPOD hydrogel group and mAMMSC composite CPOD hydrogel group according to the treatment method;(5)The wounds of each group were photographed at the corresponding time points and the remaining wound area was recorded,so as to compare the healing status of each group;(6)The fixed wound tissues were stained with H&E,so as to further observe epithelialization and collagen deposition;(7)The macrophage phenotypes of wound tissues were detected using flow cytometry;(8)Immunohistochemical staining of CD31 was carried out on the wound tissue to observe the angiogenesis of the wound.ResultsPart Ⅰ:(1)The morphology of the prepared mAM was in the form of semi-transparent quadrilateral,free of cellular and DNA components,with the size of about 400μm;(2)The phenotypic characterization of the extracted UC-MSC showed that CD105,CD90,and CD73 were positive,while CD45,CD34,CD14,CD11 b,CD19,CD79-α,and HLA-DR were negative,which met the identification criteria of MSC;(3)SEM showed that the surface of mAM was smooth and flat,while a large number of MSC could be seen on mAMMSC;(4)cytoskeletal staining results showed that the cytoskeleton of MSC on mAM was in the form of a long spindle shape,which extended throughout the entire cell;(5)live/dead staining images showed that a large number of MSC labeled with green fluorescence were attached to the surface of mAM and more MSC could be observed on mAM with the prolongation of culture time.Almost no dead cells were observed;(6)CCK-8 assay showed that the proliferation rate of mAM-MSC group was higher than that of 2D-cultured MSC,indicating that mAM could realize the rapid expansion of MSC in vitro;(7)RNA-seq results showed that compared with 2D-cultured MSC,a total of 1785 DEGs were detected in mAM-MSC group,including 773 up-regulated genes and 1012 down-regulated genes;(8)GO enrichment analysis showed that GO terms related to cell adhesion,positive regulation of cell population proliferation,extracellular matrix,cytokine activity,cytokine-mediated signaling pathway,and positive regulation of angiogenesis were significantly enriched in the mAM-MSC group;(9)KEGG enrichment analysis showed that the pathways significantly enriched in the mAM-MSC group were mainly involved in cytokine-cytokine receptor interaction,ECM-receptor interaction,and the VEGF signaling pathway;(10)The GSEA results showed that the cell-matrix adhesion,fibroblast growth factor receptor signaling pathway,positive regulation of transforming growth factor beta receptor signaling pathway,positive regulation of endothelial cell migration,blood vessel morphogenesis,blood vessel remodeling,and wound healing were all significantly activated in the mAM-MSC group,suggesting that mAM may accelerate wound healing by activating growth factor secretion,angiogenesis,and other functions in MSC;(11)The results of RT-q PCR further verified that mAM could significantly improve the growth factor secretion function of MSC.Part Ⅱ:(1)The CPOD hydrogel could crosslink at the injection site by a double-tube syringe,and the rheological detection showed that the gelation time was within a few seconds;(2)FESEM observation showed that at the microscopic level,the CPOD hydrogel presented a dense and regular 3D porous structure;(3)The elemental distribution profile showed that the mass ratio of elemental C was as high as 59.0%,and the elements of O,N,Na,and Cl respectively accounted for 29.9%,6.7%,4.0% and 0.5%,and all elements were uniformly distributed in the hydrogel;(4)The porosity of the CPOD hydrogel was 40.94 ±11.38%;(5)The degradation experiments showed that the CPOD hydrogel exhibited a slow degradation rate in saline,PBS and simulated body fluids,as long as 28 days;(6)The overlap shear strength test showed that the overlap shear strength of CPOD hydrogel was 105.45 ±1.47 k Pa;(7)The wound closure adhesion strength test showed that the wound closure strength of CPOD hydrogel was 8.07 ± 0.65 k Pa;(8)The XPS was performed in order to investigate the mechanism of the tissue adhesion of the CPOD hydrogel.Results showed amide and Schiff base bonds were generated between the interface of CPOD hydrogel and tissue;(9)Compression experiments showed the maximum compressive stress of CPOD hydrogel was 805.66 ± 33.19 k Pa;(10)Antibacterial experiments showed that the inhibition rate of CPOD hydrogel against Escherichia coli and Staphylococcus aureus was 85.52 ± 2.48%and 89.48 ± 4.12%,respectively;(11)CCK-8,live/dead staining,hemolysis experiments showed that CPOD hydrogel had better cytocompatibility;(12)The results of routine blood tests and liver and kidney function tests of mice showed that CPOD hydrogel had high biocompatibility;(13)ABTS,DPPH and PITO scavenging experiments showed CPOD hydrogel had better antioxidant capacity;(14)The results of DCFH-DA fluorescence staining showed that CPOD hydrogel could scavenge intracellular ROS.Part Ⅲ:(1)Flow analysis results showed that mAM-MSC composite CPOD hydrogel could significantly increase the proportion of M2 macrophages and inhibit the M1 polarization of macrophages,and the effect was significantly better than that of the mAMMSC group and the CPOD hydrogel group;(2)Transwell results showed that mAM-MSC composite CPOD hydrogel could significantly promote the migration of HUVECs,and the number of migrated cells was significantly higher than that of the mAM-MSC group and the CPOD hydrogel group;(3)The results of tube formation experiments showed that the mAMMSC composite CPOD hydrogel induced more formation of vascular-like structures compared with that of the CPOD hydrogel group and the mAM-MSC group;(4)Scratch experiments showed that compared with the control group,mAM-MSC composite CPOD hydrogel significantly improved the migration speed of HFB,and reached a healing rate of72.88 ± 0.50% at 16 h;(5)The results of burn wound healing experiments in C57BL/6 mice showed that the mAM-MSC composite CPOD hydrogel group achieved complete epithelialization on day 10,and the wound healing rate was significantly faster than that of the sham group,the CPOD group and the mAM-MSC group;(6)Macrophage flow analysis within the wound area showed that CPOD hydrogel(25.34 ± 0.8%),mAM-MSC(31.1 ±1.85%),and mAM-MSC composite CPOD hydrogel(35.72 ± 1.27%)transplants increased the proportion of M2 macrophages in the wound,compared with the Sham group(5.67 ±0.49%).Among them,mAM-MSC composite CPOD hydrogel had the best antiinflammatory effect with the highest proportion of M2 macrophages,which was superior to the CPOD hydrogel group and mAM-MSC group;(7)Immunohistochemical staining of CD31 showed that compared with the control group,CPOD hydrogel,mAM-MSC and mAM-MSC composite CPOD hydrogel could increase the number of blood vessels in the wound,and all of them had pro-vascularization effects,while mAM-MSC composite CPOD hydrogel had the best pro-angiogenic effect.ConclusionBased on the idea of tissue-engineered skin,mAM-MSC composite CPOD hydrogel was prepared for the treatment of burn wounds.mAM as a microcarrier,can improve the efficiency of MSC expansion in vitro,and at the same time,it can enhance the paracrine,pro-angiogenic,and wound-healing functions of MSC.mAM-MSC show great potential as seed cells in the treatment of burn wounds;The in-situ forming CPOD hydrogel has several advantages as a scaffold material,including good biocompatibility,suitable tissue adhesion ability and mechanical properties,antimicrobial properties and antioxidant properties.When CPOD hydrogel and mAM-MSC were transplanted into the burn wounds,the synergistic effect of the two ingredients could achieve better microenvironmental regulation and wound healing,including the reduction of inflammation,the promotion of vascularization,the acceleration of wound healing,and the reconstruction of tissues. |
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